Methods for making recombinant cells

ABSTRACT

Disclosed are methods for producing recombinant cells and especially recombinant mammalian cell lines with enhanced expression of an amino acid sequence. Also disclosed are recombinant mammalian cell lines producing high levels of the amino acid sequence. The methods and recombinant cell lines of the invention have a number of useful applications including use in the efficient and large-scale production of recombinant proteins and polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending application(s)application Ser. No. 09/204,979 filed on Dec. 3, 1998.

The present application is a continuation of U.S. Ser. No. 09/204,979entitled “Methods for Making Recombinant Cells” filed on Dec. 3, 1998,the disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to methods for makingrecombinant cells expressing at least one amino acid sequence. Theinvention has a variety of useful applications including use in theproduction of recombinant mammalian cells that are stable and expresshigh levels of a desired protein.

BACKGROUND OF THE INVENTION

There have been numerous attempts to produce high levels of a desiredamino acid sequence by introducing a foreign (heterologous) nucleic acidinto host cells and then expressing that nucleic acid inside the cells.Eukaryotic cells and especially mammalian cells have been employed withmixed results. For example, despite much effort toward improving methodsof making recombinant mammalian cells that produce high levels of theamino acid sequence, most of the cells do not express the nucleic acidat sufficient levels. Thus, there is a need in the field to have methodsfor generating recombinant mammalian cells that express the introducednucleic acid at high levels.

In general, two methods have been used to increase expression ofheterologous nucleic acid in mammalian cells. One approach has been toenhance nucleic acid copy number by cell selection techniques such asdrug resistance amplification. Another approach has been to increaseexpression of the nucleic acid inside the cells, e.g., by recombinantlyadding one or more a beneficial control elements to that nucleic acid.See generally Kaufman, R. J. and P. A. Sharp (1982) J Mol. Biol. 159:601; Sambrook et al. Molecular Cloning (2d ed. 1989) and Ausubel et al.Current Protocols in Molecular Biology, (19890 John Wiley & Sons, NewYork.

In particular, drug resistance amplification has been reported toinvolve cell transformation with two genes, one of which encodes anamino acid sequence of interest, such as a heterologous protein, and theother which encodes a selectable gene marker such as dihydrofolatereductase (DHFR). In instances in which the gene marker is DHFR,transformed cells are cultured in the presence of the selecting drugmethotrexate (MXT). The normally cytotoxic effects of MTX aresubstantially eliminated by expression of the DHFR. Transformed cellssurvive because they have increased (amplified) DHFR copy number to asufficiently high level. Nucleic acid sequence encoding the desiredamino acid sequence is also amplified, thereby boosting expression ofthat sequence. See e.g., Kaufman, R. J and P. A. Sharp, supra.

However, drug resistance amplification and related techniques haverecognized drawbacks. For example, generation of most recombinantmammalian cell lines using the technique is time-consuming and mayrequire several months to perform. Additionally, there has beenrecognition that when the heterologous nucleic acid is amplified,standard nucleic acid sequencing methods can be negatively impacted.Further, it is can be quite difficult to maintain sufficient nucleicacid copy number without imposing severe and sometimes long-termselection pressure. A cell selection strategy calling for extendedselection pressure can be expensive and may present regulatory issuessuch as when a heterologous protein is being produced for pharmaceuticaluse.

In addition, drug resistance amplification may not be able to enhanceexpression of specific heterologous nucleic acids to levels sufficientfor applications such as commercial or research use.

Further problems with drug resistance amplification include geneticinstability of cloned cell lines. These problems are highly significant.For example, the method often produces recombinant cells that evolvefrom unstable gene amplification events, e.g., formation of doubleminute chromosomes. These cells can lose desired characteristics whenthe selecting drug is removed. See Kaufman, R. J. et al. (1985) Mol.Cell. Biol. 5: 1750 and references cited therein.

Additionally, optimal practice of most drug resistance amplificationtechniques has required use of highly specialized cells, vectors and/orcell growth conditions. For example, most of the methods employ hostcells that have been genetically manipulated in specific ways. Suchmutant cells may not be suited for some applications. As an illustrationof the difficulties, drug resistance amplification involving DHFR andMTX will not work optimally in cells carrying a normal DHFR gene.Disabling that normal gene can be a lengthy and laborious process. Seee.g., Wigler et al. (1980) PNAS (USA) 3567; and Urlaub and Chasin,(1980) PNAS (USA) 77: 4216.

More particular drawbacks of DHFR/MTX amplification methods relating touse of specific vectors and growth conditions have been disclosed. Seee.g., Kaufman and Sharp, supra; Schimke, R. Cell (1984) 37:705. See alsoU.S. Pat. Nos. 5, 686,263; 4,956,288 and 5,585,237 and references citedtherein.

It would be useful to have methods for making recombinant cells that areflexible and can be used to produce recombinant cell lines that aregenetically stable. It would be particularly desirable to have methodsfor producing recombinant mammalian cell lines that significantly reduceor avoid use of highly specialized cells, vectors and/or growthconditions. It would be further desirable to have recombinant mammaliancell lines made by those methods that produce high levels of an aminoacid sequence of interest such as a heterologous protein.

SUMMARY OF THE INVENTION

The present invention relates to methods for producing recombinant cellsand particularly to methods for producing recombinant cell lines thatare genetically stable and express high levels of at least one aminoacid sequence. In one aspect, the invention provides methods forproducing recombinant cell lines that express high levels of aheterologous protein. The methods generally involve introducing intohost cells at least one vector encoding the amino acid sequence andsubjecting the cells to conditions conducive to isolating recombinantcell lines that produce high levels of that sequence. Preferred methodsproduce high levels of the amino acid sequence while significantlyreducing use of highly specialized host cells, vectors and/or growthconditions. Additionally provided are recombinant mammalian cell linesproduced by the methods of this invention.

The present invention more particularly relates to methods forgenerating recombinant mammalian cell lines that are genetically stableand express high levels of at least one amino acid sequence of interest.In one embodiment, expression of the amino acid sequence issubstantially enhanced by introducing into host cells a first vectorencoding the sequence. The first vector includes at least one selectablesequence, typically a first selectable sequence, operably linked to asegment encoding the amino acid sequence. Additionally, the first vectormay encode more than copy of the amino acid sequence if desired. Thefirst vector can be introduced into the host cells once (singly) or morethan once (multiply) as needed. The host cells are then cultured underconditions conducive to expressing the amino acid sequence followed byisolation of recombinant cell lines (first high expressing cells)expressing the sequence at a first high expression level.

The method further includes introducing, preferably into the first highexpressing cells, a second vector encoding the amino acid sequence. Thesecond vector may be the same or different from the first vector and itmay encode more than one copy of the amino acid sequence if needed. Thesecond vector is introduced into the first high expressing cells once(singly) or more than once (multiply) as needed. Recombinant cellscomprising the second vector are then cultured under conditionsconducive to expressing the amino acid sequence. Cells expressing theamino acid sequence at a second high expression level (second highexpressing cells) are subsequently isolated.

As discussed, there has been recognition that cell lines made by priorcell selection schemes and especially drug resistance amplificationtechniques have suffered from drawbacks. Specific drawbacks includegenetic instability and the need to use highly specialized cells,vectors and/or growth conditions. The present methods significantlyavoid these drawbacks by providing genetically stable cell lines thatproduce high levels of the amino acid sequence. Further, the presentmethods are generally more flexible than the prior schemes and arecompatible with a wide spectrum of vectors. Nearly any transfectablecell can be used with the present methods including most primary,secondary or cultured mammalian cells. Suitably, the present methodsprovide for growth of a specific transfectable mammalian cell under avariety of selective or nonselective growth conditions as needed.

More particular flexibility is provided by providing for either director indirect selection of recombinant cell lines expressing high levelsof the amino acid sequence. For example, in one embodiment of thepresent methods, recombinant mammalian cell lines are selectedindirectly by selection of at least one vector-encoded cell surfacemarker. Selection of that cell surface marker in accord with theinvention facilitates efficient isolation of cell lines that express theamino acid sequence at high levels. As discussed, resulting cell linesare genetically stable and can be produced while minimizing oreliminating use of highly specialized cells, vectors, and/or growthconditions.

In a particular embodiment of the method, the second high expressionlevel is substantially higher than the first high expression level by atleast from about 2 to 10 fold higher or more. Methods for determininglevels of amino acid sequences such as proteins are known in the fieldand include certain techniques such as antibody reactivity.

In a more particular embodiment, the method further includes introducinginto the second high expressing cells, a third vector encoding the aminoacid sequence and subjecting the cells to conditions conducive toexpressing the amino acid sequence at a third expression level higherthan the second expression level. Cells expressing the amino acidsequence at the third expression level are then isolated to produce thecell line (third high expressing cells). The third vector preferablyencodes at least one amino acid sequence of interest and it can be thesame or different from the first or second vector (or both vectors). Thethird vector can be introduced into the second high expressing cellsonce (singly) or more than once (multiply) as needed. The third highexpressing cells will typically exhibit increased expression of theamino acid sequence when compared to the first or second high expressingcells.

In another particular embodiment, the method further includesintroducing into the third high expressing cells at least one vectorencoding the amino acid sequence, preferably one of such vectors, andsubjecting the cells to conditions conducive to expressing the aminoacid sequence at a level higher than the third expression level. Themethod further includes repeating the introduction and subjecting stepsat least once, preferably from between about 2 to 10 times or more, toisolate a cell line expressing the amino acid sequence at a higher levelthan the third high expressing cells. Choice of how many times the stepsare repeated will be guided by several parameters but particularly bythe level of expression required.

In another embodiment, the step of culturing the host cells underconditions conducive to selecting the first vector further includesgrowing the host cells in selective media. Preferably, that selectivemedia includes at least one drug and usually one drug selective for thefirst selectable sequence. It will be appreciated that vectors disclosedherein that include a selectable nucleic acid sequence, e.g., the firstand fourth vectors, usually encode an amino acid sequence that isselectable. Illustrative drugs for selecting the vectors are describedin the discussion and examples that follow.

It will often be preferred that the second or third vector (or both ofthe vectors) be co-introduced into cells with at least one other vector(sometimes referred to herein as “fourth” or “fifth” vector), whichvector encodes at least one selectable sequence and particularly aselectable cell surface marker such as a cell surface protein. It hasbeen surprisingly found that by co-introducing the vector encoding theselectable cell surface marker, it is possible to facilitate productionof highly useful recombinant mammalian cell lines expressing high levelsof the desired amino acid sequence. For example, it has been found thatby co-introducing that vector in accord with the invention, it ispossible to generate cell lines while reducing or totally eliminatingthe need to use highly specialized cells, vectors and/or growthconditions. Additionally, production of genetically stable recombinantcells is favored by practice of the methods.

In another embodiment of the method, the step of introducing the secondvector into the first high expressing cells further comprisesintroducing the fourth vector into the cells. As discussed, the fourthvector encodes at least one selectable sequence (referred to herein asthe “second” selectable sequence) preferably a selectable cell surfacemarker. The fourth vector may be the same as another vector of themethod although in most cases the fourth vector will be different fromany one or all of these vectors. In another embodiment, the fourthvector comprises a second selectable sequence operably linked to asegment encoding the amino acid sequence of interest. In a more specificembodiment, the method further includes growing the cells in selectivemedia including at least one drug selective for the second selectablesequence.

In embodiments in which the fourth vector encodes the cell surfacemarker, the method preferably further includes isolating the cellsexpressing that selectable cell surface marker by at least one ofchromatography, cell panning, flow cytometry, antibody binding,immunoprecipitation, or antibody binding. Usually one of thesetechniques will be employed to isolate the cells expressing the cellsurface marker. By conducting the isolation, cell lines expressing highlevels of the amino acid sequence encoded by the vectors is obtained.

In another embodiment of the method, the step of introducing the thirdvector into the second high expressing cells further includesintroducing into the cells a fifth vector. In a particular embodiment,the fifth vector encodes at least one selectable sequence (referred toherein as the “third” selectable sequence). The fifth vector may be thesame or different from any of the vectors disclosed herein. In a moreparticular embodiment, the fifth vector includes a third selectablesequence which if desired can be operably linked to a segment encodingthe amino acid sequence. The cells are then subjected to conditionsconducive to expressing the amino acid sequence at a third expressionlevel higher than the second expression level. Preferably, the methodfurther comprises growing the cells in selective media that includes atleast one drug and preferably one drug selective for the thirdselectable sequence.

An additionally preferred fifth vector further includes a sequenceencoding a selectable cell surface marker which can be the same ordifferent from the cell surface marker encoded by the fourth vector. Ifdesired, the selectable cell surface marker can be operably linked to asegment encoding the amino acid sequence. The method preferably furtherincludes the step of isolating the cells expressing the selectable cellsurface marker encoded by the fifth vector and using at least one ofchromatography, cell panning, flow cytometry, antibody binding,immunoprecipitation, or antibody binding to isolate cells expressing thecell surface marker expressed by the fourth or fifth vector (or bothvectors) as needed. By performing the isolation, recombinant cell linesexpressing high levels of the amino acid sequence encoded by the vectorsis obtained.

More particular second high expressing cells in accord with theinvention generally express at least about 2 fold more of the amino acidsequence than the first high expressing cells as determined by standardprotein quantitation techniques such as quantitative gelelectrophoresis, chromatography and immunological techniques such asWestern immunoblot, ELISA and antibody reactivity. More particularsecond high expressing cells express from between about 3 to about 40fold of the amino acid sequence when compared to the first highexpressing cells as determined by antibody reactivity.

Third high expressing cells of particular interest generally express atleast about 2 fold more of the amino acid sequence than the second highexpressing cells as determined by the standard protein sequencequantitation techniques. More particular third high expressing cellsexpress from between about 3 to about 40 fold of the amino acid sequencewhen compared to the second high expressing cells as determined byantibody reactivity.

In a more specific embodiment of the methods, each of the first, secondor third vectors independently encodes a first drug resistance gene(sometimes referred to herein as a gene marker), a selectable cellsurface marker and a second drug resistance gene, respectively. In thisembodiment, each of the vectors is operably linked to a segment encodingthe amino acid sequence of interest. Additionally, the fourth and fifthvectors can each independently encode a third drug resistance gene andanother cell surface marker, respectively. The first, second and thirddrug resistance genes can be the same or different as needed.Illustrative drug resistance genes and selectable cell surface markersare described in more detail below.

As an illustration of the invention, a suitable mammalian host cell isfirst selected that does not require highly specialized vectors orgrowth conditions to optimally express a heterologous protein. Preferredmammalian host cells do not express the heterologous protein atdetectable levels. However in some instances, suitable mammalian hostcells may already express the protein as a homologous protein, e.g., atbackground (ie. basal) levels. In this instance, the invention can beused to boost expression of the homologous protein to levels higher thanthe basal levels. Alternatively, the present methods can be used toboost cell expression of a desired heterologous protein or polypeptidesequence. More specific mammalian host cells are provided below.

Following introduction of the first vector into the mammalian hostcells, the first high expressing cells are isolated in growth media thatis generally selective for the first vector. Second high expressingcells are produced by introducing the second vector encoding the aminoacid sequence into the first high expressing cells. In one embodiment,the first and second vectors encode one copy of a heterologous proteinand are substantially the same. Introduction of the second vector isaccompanied by introduction of another (“fourth”) vector encoding theselectable cell surface marker. Isolation of the second high expressingcells is performed under conditions conducive to selecting that cellsurface marker and may optionally include growth in selective media ifdesired. In this embodiment, selection of the cell surface marker alsoselects for high coexpression of the desired amino acid sequence encodedby the first and second vectors. Use of highly specialized host cells,vectors and growth media to select the cells is significantly reduced inthis example.

The present invention can be used to make a wide spectrum of recombinantcells and particularly mammalian cell lines that express high levels ofat least one desired amino acid sequence of interest or a portion ofthat amino acid sequence such as a functional protein fragment. Forexample, methods of the invention can be used to express amino acidsequences of immunological interest such as an immunoglobin chain (heavyor light) or functional fragments thereof. More specific amino acidsequences of interest are discussed below.

The present invention provides significant advantages when contrastedwith prior cell selection techniques and especially conventional drugresistance amplification methods. For example, as discussed, practice ofmost drug resistance amplification methods generally requires use highlyspecialized cell lines, vectors and growth conditions. In mostinstances, the cells have been genetically manipulated to blockresistance to a drug and may not always exhibit good growthcharacteristics. Further, such cells can be difficult to make and/ormaintain and may be particularly unsuitable for many specific cellselection strategies. The present invention addresses these shortcomingsby providing methods for generating recombinant cell lines that reduceor eliminate use of these highly specialized cell lines.

Further, the present methods maximize the genetic stability of resultingrecombinant cell lines by introducing heterologous nucleic acids intothe host cell genome. Formation of highly unstable chromosome formationsis reduced and often totally avoided.

Additional advantages are provided by this invention. For example, thepresent methods are substantially more flexible than prior methods forgenerating recombinant mammalian cells and can be used with a variety ofmammalian expression vectors. In contrast, most prior cell selectiontechniques such as drug resistance amplification are tailored for usewith highly specialized vectors. As an illustration of the problem,there has been acknowledgement that vectors for many drug resistanceamplification techniques have become too large and difficult tomanipulate. Also many optimal vectors for use with prior cell selectionschemes are proprietary and may not always be available when needed. Thepresent invention solves this problem, e.g., by providing compatibilitywith a wide variety of mammalian expression vectors.

As discussed, at least one of the vectors described herein may encode aportion of the amino acid sequence of interest. As an illustration, fora specific full-length heterologous protein, the first vector can encodea first portion of that protein and at least one of the second, third,fourth and fifth vectors can encode other portions of that protein thesame or different. In this example, the totality of the protein sequenceencoded by the vectors will be substantially equivalent to thefull-length protein. In another example, the first vector (or any otherof the vectors) may encode the full-length and at least one other of thevectors, e.g., the second, third or fourth vector may encode a specificportion of the protein. The remaining vectors can be used, e.g., tointroduce additional portions of the protein sequence or even thefull-length protein sequence if desired.

The ability of the present methods to introduce portions of an aminoacid sequence of interest provides advantages. For example, the methodscan be used to provide highly controlled amplification of one or severalportions of the amino acid sequence. That is, specific portions of theamino acid sequence including the entire sequence can be amplified inspecific cells (e.g., first or second high expressing cells) prior to orconcurrent with amplification of another sequence. The inventiontherefore provides for sequential and coordinate expression of one oreven several amino acid sequences of interest.

As a more specific illustration, the first vector can encode a firstsubunit of a multi-subunit protein (homologous or heterologous) and thesecond vector can encode a second subunit of that same protein. In thisexample, first vector encoding the first subunit can be introduced intothe host cells and the first high expressing cells selected. The secondvector encoding the second subunit can be introduced into the first highexpressing cells and the second high expressing cells selected thatexpress both the first and second subunits. If, for example, themulti-subunit protein is a dimer, that protein can be specificallyformed in the first high expressing cells under conditions of enhancedexpression of the first and second subunits. Thus, the present inventionis especially useful for analyzing assembly and stability ofmulti-subunit proteins under conditions of sequential and highlycontrolled subunit expression.

Particularly contemplated is sequential and highly controlled assemblyof proteins of immunological interest such as immunoglobins andparticularly immunoglobin heavy and light chains.

More generally, the present invention facilitates implementation ofstrategies for assembling the multi-subunit proteins by providingsubstantial flexibility and control over the amplification process. Forexample, the methods can be used to enhance production of the subunits,e.g., at pre-determined high expression levels inside cells. Incontrast, most prior techniques and particularly drug resistanceamplification techniques are substantially less flexible and do notprovide control over the subunit assembly.

In another aspect, the present invention provides cell lines andparticularly recombinant mammalian cell lines that are produced by themethods disclosed herein. Such cell lines are genetically stable and arespecifically selected to express the amino acid sequence or portionthereof at high levels. The methods of this invention specificallyprovide for selection of recombinant mammalian cell lines that expressat least heterologous protein at high levels.

Particular methods of this invention provide additional advantages. Forexample, practice of methods to generate recombinant mammalian celllines (sometimes called “high producing cell lines” or related term) canbe achieved in significantly less time than other cell selection methodsincluding most prior drug amplification methods.

All documents disclosed herein are incorporated by reference in theirentirety. The following non-limiting examples are illustrative of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are drawings showing illustrative vectors for use withthe invention. (1A) vector pJAIgG4TF encodes a chimeric anti-tissuefactor (TF) heavy and light immunoglobin chains. (1B) vector pDRHKencodes a single-chain HLA-DR2/MBP molecule fused to a humanimmunoglobin kappa constant domain.

FIG. 2 is a graph showing high level production of recombinant chimericanti-TF antibody in T25 flask static cultures. Recombinant cell linesproducing high levels of the anti-TF antibody were isolated followingthree sequential transfections. High producing recombinant cell lineswere designated H9G12, 3D2A9, and A11B5, respectively.

FIG. 3 is a graph showing high level production of recombinant chimericanti-TF in a bioreactor. Recombinant cell lines producing high levels ofthe anti-TF antibody were isolated following three sequentialtransfections. High producing recombinant cell lines were designatedH9G12 and A11B5 for lines isolated after the first and thirdtransfections.

FIG. 4 is a graph showing high level production of recombinantsc-DR2/MBPCκ production in T25 flask static cultures. Recombinant celllines producing high levels of the sc-DR2/MBP-C_(κ) fusion protein wereisolated following three sequential transfections. High producingrecombinant cell lines were designated A5B4, DR2H4, and B9 respectively.

DETAILED DESCRIPTION OF THE INVENTION

As summarized above, the present invention provides methods for making awide spectrum of cells and particularly recombinant cell lines.Particularly provided are methods for making recombinant mammalian celllines that are genetically stable and produce high levels of aheterologous or homologous protein of interest. As discussed, theinvention is flexible and can be used to make highly useful cell linesthat produce high levels of the protein while significantly avoiding useof highly specialized cells, vectors and/or growth conditions.Additionally provided are recombinant mammalian cell lines that producehigh levels of the protein.

In general, optimal practice of the present invention is achieved by useof recognized manipulations. For example, techniques for isolating DNA,making and selecting vectors for expressing the DNA, purifyng andanalyzing nucleic acids, specific methods for making recombinant vectorDNA, cleaving DNA with restriction enzymes, ligating DNA, introducingDNA including vector DNA into host cells by stable or transient means,culturing the host cells in selective or non-selective media, exemplaryhost cells methods for selecting and maintaining cells stably ortransiently expressing the DNA, are generally known in the field. Seegenerally Sambrook et al., supra; and Ausubel et al., supra.

The present invention provides, in one aspect, novel methods forgenerating recombinant mammalian cell lines. A recombinant mammaliancell line will sometimes be referred to herein as a “high producing”cell line or related term. The phrase “high level”, “high producing” orthe like, when used to reference the amount of an amino acid sequenceproduced by the cell line, means that at least about 2 fold andpreferably from between about 3 to about 40 fold or higher (someheterologous sequences are not present in the parental cell line) moreof the amino acid sequence is produced by the cell line compared to aparental cell or parental cell line.

Methods for determining the amount of a particular amino acid sequenceproduced by a recombinant cell line described herein are known in thefield and include chromatographic methods such as protein gelelectrophoresis and immunological techniques such as Western blottingand ELISA. More particular gel electrophoretic methods include those inwhich a protein or peptide sequence is detected and quantitated usingCoumassie blue or silver staining. A preferred protein quantitationmethod is antibody reactivity.

By the term “antibody reactivity” is meant specific binding between adesignated antibody and the amino acid sequence produced by the cell orcell line. The term is further meant to reference formation of aspecific binding pair between the amino acid sequence (ie. an epitopethereon) and the antibody but which does not significantly bind to othermolecules as determined, e.g., by Western blotting, ELISA, RIA, gelmobility shift assay, enzyme immunoassay, competitive assays, saturationassays or other suitable amino acid sequence binding assays known in thefield. See generally, Harlow and Lane in Antibodies: A LaboratoryManual, CSH Publications, N.Y. (1988), for disclosure relating to theseand other suitable assays.

By the term “parental” as it is used to reference a host cell or cellline is meant an ancestor of that cell or cell line used to make asubsequent cell line. Illustrative parental cells are suitable hostcells used to make the first high expressing cells. The first highexpressing cells are in turn an example of a parental cell line used tomake the second high expressing cells. It will be understood that a cellline is a clonal population of cells derived form a single ancestor cellunless otherwise stated. Methods for making cell lines are generallyknown in the field and include well-known serial dilution techniques.See e.g., Ausubel et al., supra.

In accord with one aspect of the invention, mammalian host cellsproviding enhanced expression of a specific amino acid sequence such asa heterologous protein are obtained by introducing into the cells afirst vector that includes at least one selectable sequence andpreferably one selectable sequence (first selectable sequence) operablylinked to the segment encoding the protein. Following culturing andisolation of cells expressing the protein at the first high expressionlevel (first high expressing cells), a second vector encoding theprotein is introduced into the first high expressing cells. It isgenerally preferred that the introduction of the second vector beaccompanied by introduction of another vector (ie. fourth vector) whichencodes at least one and preferably one selectable cell surface marker.However, in other embodiments, the second vector encodes both theprotein and at least one cell surface marker, preferably one of suchmarkers. The cells are subjected to conditions conducive to expressingthe amino acid sequence at a second expression level that is higher thanthe first expression level. Preferred conditions select for at least oneof the cell surface markers and do not use highly specialized cells,vectors and growth conditions. A cell line expressing the protein at thesecond expression level is then isolated to produce the second highexpressing cells.

By the term “isolated” as it refers to specific cells or cell linesdisclosed herein means cells that have been purified to at least about80 to 95% (w/w) homogeneity. Purified cells of higher purity, e.g., atleast about 98% to 99% homogeneity (w/w) are more preferred for mostapplications. Once purified, the cells can be used for subsequentmanipulations such as cell transfections or establishment of cell linesusing recognized serial dilution techniques.

As discussed, the first and/or second vectors can be introduced intorespective cells once (singly) or more than once (multiply), preferablyfrom between about 2 to about 20 times and more preferably from betweenabout 2 to 5 times, as needed. Choice of whether to introduce the firstand/or second vector into respective cells once or more than once willbe guided by several parameters such as the level of enhanced expressiondesired and the particular amino acid sequence of interest.

In a more particular embodiment, the conditions used to select thesecond high expressing cells include selection of at least one cellsurface marker and preferably one cell surface marker, which marker isencoded by one of the vectors co-introduced into the first highexpressing cells. As discussed, those vectors can be the fourth or fifthvector. It is emphasized that this embodiment of the present methods isa substantial improvement over prior expression systems such as drugresistance amplification techniques. For example, by co-introducing thevectors into the cells, selection of the second high expressing cellscan be conducted by selection of the cell surface marker, thereby manyof the problems already discussed. Also significantly, sequentialintroduction of the first and second vector in accord with thisinvention can enhance levels of expression of the amino acid sequenceabove the sum of the levels of the amino acid sequence.

An “amino acid sequence” generally refers to any polymer consistingessentially of any of the 20 amino acids regardless of its size.Although the term is used herein to reference proteins, the term will beunderstood to encompass polypeptides and peptides unless specificallystated otherwise. The amino acid sequence may encode a full-lengthprotein (heterologous or homologous with respect to the expressing cell)or a portion thereof such as a functional fragment. More specific aminoacid sequences are described below. A specifically preferred amino acidsequence is an immunoglobin heavy chain, light chain; or a functionalfragment thereof.

By the term “functional fragment” as it is used with respect to an aminoacid sequence is meant at least a segment of an amino acid sequence thathas at least one of the activities of the full-length amino acidsequence. With respect to protein sequences, those activities includespecific binding and enzymatic activity. Preferred functional fragmentshave at least about 70% and more preferably from between about 80% to95% of the activity of the full-length protein as determined by asuitable assay.

By the term “operably linked” is meant linkage of polynucleotideelements in a functional relationship. A nucleic acid is “operablylinked” in accord with this invention when it is placed into afunctional relationship with another nucleic acid sequence. Inparticular, operably linked sequences may reside on the same vector oron different vectors within the same cell. For instance, a promoter orenhancer is operably linked to a specific coding sequence if it affectstranscription of that coding sequence. That is, for this example,operably linked means that the DNA sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin reading frame. However, operably linked sequences may reside ondifferent vectors in some instances. As an illustration, a vectorsequence encoding one subunit of a multi-subunit protein would beoperably linked to another vector sequence encoding another subunit(binding partner) of that protein.

Methods of the present invention are operable and highly useful with awide spectrum of host cells, particularly those well adapted to tissueculture. Typically, the host cells are eukaryotic, preferably mammaliancells that exhibit good growth characteristics in standard mediapreparations. Substantially, nearly any non-microbial cell, whether ornot previously transformed, and which can express a desired amino acidsequence at high levels can be used in accord with the invention. Avariety of such cells are known in the art and include CHO, CV-1, HeLaand other cells. See generally Sambrook et al., supra and Ausubel etal., supra; and the American Tissue Type Culture Collection, 10801University Boulevard, Manassas, Va.

As will become even more apparent from the discussion and examples whichfollow, a variety of vectors and especially mammalian expression vectorscan be used with the present invention. Particular vectors willgenerally include suitable regulatory sequences for self-replication andpreferably selection in a desired host cell or cell line.

The term “vector” is more particularly defined as any nucleic acidsequence of interest capable of being incorporated into a host cell andresulting in the expression of a nucleic acid sequence of interest. Thatnucleic acid sequence of interest will preferably encode all or asubstantial part of the amino acid sequence described above. Suitablevectors can include but are not limited to linear nucleic acidsequences, plasmids, cosmids, phagemids, episomes and extrachromosomalDNA. Additionally included are viral DNA and viral RNA. Specifically,the vector can be a recombinant DNA. Also used herein, the term“expression” or “gene expression”, is meant to refer to the productionof the protein product of the nucleic acid sequence of interest,including transcription of the DNA and translation of the RNAtranscript.

More particular vectors for use with the present invention include atleast one selectable nucleic acid sequence, usually one of such asequence. For purposes of illustration, selectable sequences aresometimes referred to as a selectable gene marker (or related term) whenthe sequence encodes an intracellular sequence; or cell surface marker(or related term) when the sequence encodes a cell surface protein.

It will be apparent that a wide variety of vector-encoded selectablesequences are compatible with the invention. Preferred vectorsfacilitate selection of host cells or cell lines that harbor the vectoror other vectors co-introduced in the cell. That goal can be achieved bya variety of techniques including substantial survival of the cellsfollowing exposure to a cytotoxic drug. See generally, Southern, P. J.et al. (1982) J. Mol. AppL. Gen. 1: 327; Sambrook et al. supra; andAusubel et al. supra..

For example, illustrative selectable gene markers for use with thisinvention include DHFR, aminoglycoside antibiotic G418, hygromycin B(hmb), and neomycin phosphotransferase II gene (neo), puromycin and theadenosine deaminase gene for certain auxotrophic eukarytotic cells,e.g., Chinese hamster ovary (CHO) cells. It is contemplated that in somesituations, the desired amino acid sequence encoded by the vector mayitself be a sufficient selectable marker or may positively impactfunction of the selectable marker. In cases in which the amino acidsequence itself encodes the selectable marker, a separate selectablemarker need not be included in the vector. See e.g., Southern, P. J. etal. (1981) J Mol Biol 150:1; Sambrook et al., supra; and Ausubel et al.supra and references therein for additional disclosure relating to genemarkers.

As discussed, the selectable nucleic acid sequence may encode a suitablecell surface marker and particularly a cell surface protein. Exemplarycell surface markers include a cell surface receptor, glycoprotein,carbohydrate, protein, lipoprotein, major histocompatibility complex(MHC/HLA), antibody, antigen or a functional fragment thereof. Moreparticular cell surface markers include glycoproteins of immunologicalinterest such as CD (cluster of differentiation) glycoproteins typicallyfound on T-cell surfaces. Examples include CD2, CD3, CD4, CD8 and LFA-1.A more particular glycoprotein of interest is the CD4 molecule andespecially a functional fragment of that molecule. See also theCaptureTec™ System provided by InVitrogen.

As discussed above in the examples that follow, cells that express aspecific cell surface marker can be isolated by one or a combination ofdifferent strategies. For example, in one approach, the cells can beisolated by chromatography, cell panning, flow cytometry, antibodybinding or immunoprecipitation. A preferred approach is chromatographyinvolving magnetic selection as discussed below.

By the term “functional fragment” as the term is used to define aselectable cell surface marker is meant a portion of that marker, e.g.,the CD4 glycoprotein, which is capable of specifically binding anotherimmunological molecule such as an antibody like a monoclonal antibody.The term is also meant to include nucleic acid sequence encoding thefunctional fragment.

In general, once a suitable selectable sequence is chosen, it will beoperably linked to one or more regulatory sequences so that the encodedamino acid sequence is positioned in the vector, e.g., adjacent to apromoter. In this way, expression of the desired amino acid sequence isfacilitated. It is contemplated that some amino acid sequences such as aheterologous or homologous protein will have its own cell or tissuespecific promoter. Further included regulatory sequences are suitableleader and polyadenylation sequences.

In more particular examples of the present invention, expression of theamino acid sequence is either as a mono- or di-cistron as needed. Forexample, expression is monocistronic when the promoter is operablylinked to both the selectable nucleic acid sequence and the amino acidsequence of interest, e.g., promoter/encoded amino acidsequence/selectable nucleic acid sequence; promoter/selectable nucleicacid sequence/encoded amino acid sequence. Alternatively, dicistronicexpression follows from expressing the amino acid sequence from separatepromoters, e.g., promoter/encoded amino acid sequence or selectablenucleic acid sequence.

As discussed, the present methods are compatible with a wide variety ofvectors. These vectors typically encode the amino acid sequence (orseveral of such sequences) for which enhanced expression is desired. Apreferred vector format is sometimes referred to as expression cassette.In one embodiment, the expression cassette generally includes a promoterfunctional in the cell or cell line hosting the vector, an operator, aribosomal initiation site, the amino acid sequence, and an sufficient 3′portion encoding polyadenylation signals to facilitate processing and,in some cases, secretion of the amino acid sequence from the cell. Apreferred example of a terminator sequence is the polyadenylationsequence from SV40. If desired, the expression cassette or otherappropriate portion of the vector may include a signal sequence near the5′ end of the amino acid sequence to facilitate post-translationalprocessing of that sequence. At least one suitable gene marker or cellsurface vector can be positioned in the vector if needed.

A more specific example of a suitable vector including the expressioncassette is a DNA vector comprising (i) an origin of replication (Ori)functional in E. coli; (ii) a selectable gene marker (antibioticresistance gene e.g., Amp, Tet, Neo or Kan resistance); (iii) a strongviral promoter such as the cytomeglovirus (CMV) promoter and optionalCMV enhancer element, (iv) an (Ig-C_(L)) immunoglobin light chainconstant region leader sequence, (v) the amino acid sequence ofinterest, (vi) a full-length Ig-C_(L) intron linked to an Ig-C_(L) exon,(vii) a growth hormone polyadenlyation sequence, e.g., bovine growthhormone (bgh) poly A sequence and (viii) DNA encoding a selectableeukaryotic marker such as a strong viral promoter (e.g., simian virus 40(SV40) promoter) linked to the antibiotic resistance gene and fused to aviral polyadenlyation sequence (e.g., the SV40 polyA sequence).Alternatively, the DNA vector can include all of (i)-(v), and(vii)-(viii), above, without the full-length Ig-C_(L) intron linked tothe Ig-C_(L) exon of (vi). An exemplary Ig-C_(L) leader sequence is themouse kappa leader. An example of a fill-length Ig-C_(L) intron and exonis the full-length Cκ gene.

The amino acid sequence for which enhanced expression is desired can benearly any protein or polypeptide sequence including heterologousproteins or homologous (endogenous) proteins naturally produced by thehost cell. In most cases, the amino acid sequence will be a eukaryoticprotein including, but not limited to, a subunit or functional fragmentof a larger amino acid sequence such as a multi-subunit protein. Morespecific amino acid sequences include enzymes, immunoglobulins, peptidehormones, vaccines, receptors, including T-cell receptors, MHC/HLAmolecules (class I and class II); or fragments thereof. Additionallyspecific proteins include growth factors, blood coagulation factors,cytokines, e.g., plasminogen activator, tissue factor (TF), insulin,mammalian growth hormone, erythropietin, IgE, urokinase, interleukins 1,2, and 3; or fragments thereof. The invention is further compatible withother known, partially known, or unknown amino acid sequences includingthose encoding novel gene sequences.

A variety of such amino acid sequences can be found, e.g., in Genbank(National Library of Medicine, 38A, 8N05, Rockville Pike, Bethesda, Md.20894). Genbank is also available on the internet athttp://www.ncbi.nlm.nih.gov.

Molecular weights of specific vectors discussed herein can be determinedby conventional techniques such as agarose gel electrophoresis sizingand will vary depending, e.g, on intended use. However, most suitablevectors and especially suitable DNA vector will have a molecular weightof at least about 5 kb and particularly from between about 5 kb to about35 kb or higher. The molecular weight of particular amino acid sequencescan be determined by standard protein sizing techniques such aspolyacrylamide gel electrophoresis. Alternatively, or in addition, themolecular weight can be estimated by determining the molecular weight ofthe corresponding nucleic acid sequence followed by conceptualtranslation of that sequence. For most applications, the size of thenucleic acid encoding the amino acid sequence will be sufficient toencode a protein or polypeptide of from between about 500 to about300,000 daltons with about 15 to about 200,000 daltons being generallypreferred.

See the following examples for more specific vectors for use with thisinvention such as pJAIgG4Tf.A8, pDRHK, and pMACS. See also FIGS. 1A and1B. In particular, the pDRHK vector has been deposited pursuant to theBudapest Treaty with the ATCC at the address disclosed above. The DNAvector was deposited with the ATCC on Sep. 17, 1997 and was assignedAccession No. 209274. The pDRHK vector is a mammalian expression vectorwhich includes a CMV promoter, mouse IgC kappa leader peptide, cloningregion, mouse kappa intron and human kappa constant domain exonsequence.

As discussed above, the present invention features a series ofrecombinant manipulations involving sequential and co-ordinate vectorintroduction to generate recombinant mammalian cell lines that expresshigh levels of an amino acid sequence. Introduction of the vectors canbe achieved by a variety of ways including retroviral transfer, viralinfection, calcium-, liposome-, or polybrene- mediated transfection,biolistic transfer, or other such techniques known in the art. Morepreferred introduction methods include those adaptable for stabletransfection of mammalian cells such as electroporation. However, forsome applications it may be useful to employ at some transientintroduction methods provided the resulting recombinant cell lines aregenetically stable.

As a more specific illustration of the invention, a suitable mammalianhost cell such as CHO is transfected once (singly) or at least twice(multiply) with suitable vectors each including at least one selectablemarker and encoding at least one heterologous protein of interest. In amore specific embodiment, the host cells are transfected with one vectorthat includes a first selectable sequence (gene marker) operably linkedto a segment operably linked to the encoded protein. A variety oftransfection methods can be used to introduce the vector into the hostcells such as standard calcium phosphate mediated transfection orlipofection techniques. Transfected host cells are then subjected toselective growth conditions so that first high expressing cells can beisolated. Methods for isolating transfected cells have been described ine.g., Sambrook et al., supra; Ausubel et al. supra; and in Wigler PNAS(USA) (1979) 76: 1376.

The first high expressing cells are isolated and can be characterized ifdesired by one or a combination of standard techniques. For example, inone approach, the CHO cells are transfected with a vector encoding aneomycin gene marker (providing G418 resistance) operably linked to aprotein sequence such as an inmmunoglobin-encoding sequence andparticularly a sequence encoding an antibody. Transfected host cellsexpressing the protein sequence are then cloned by standard techniques(e.g., limiting dilution and grow-up) performed in microtitre tissueculture plates. After incubating the cells for about a few days up to afew weeks or longer, single colonies will typically appear. Preferablyonly single colonies are used for additional manipulations. Highproducing clones are selected by any acceptable method such as standardimmunological methods and particularly an ELISA assay. Preferred areELISA assays optimized to detect and quantitate antibody production.Preferred high producing cells are those that produce substantially thehighest amount of antibody in the ELISA assay. More preferred are firsthigh producing cells producing from between about 0.5 to about 20micrograms antibody per milliliters of media. Specifically preferred arefirst high producing cells producing from between about 1 to about 5micrograms antibody per milliliters of media.

The first high producing cells are further manipulated particularly toincrease cell copies of nucleic acid encoding the protein sequence. Aspecific goal in this example is to produce a genetically stableantibody producing cell line. In one embodiment, the first highproducing cells are further transfected, e.g., by electroporation, withtwo different vectors: one encoding the protein sequence and the othervector encoding a suitable cell surface marker such as a glycoproteinand especially CD4 or a fragment thereof. See FIG. 1A. A preferredcommercially available vector encoding the CD4 is pMACS (see below). Inthis example, transfectants are treated with a specific antibody orsuitable antigen binding fragment thereof capable of specificallybinding the cell surface protein.

By the term “genetically stable” as it is meant herein to refer to acell means a cell that is substantially free of genomic rearrangementsthat include the heterologous nucleic acid. Particularly avoidedrearrangements include double-minute chromosomes. The present methodspreferably introduce the heterologous nucleic acids into the hostingcell genome to maximize genetic stability.

The specific binding between cells expressing the cell surface proteinfrom one of the vectors and the protein sequence encoded by the othervector can be detected in a variety of ways including standardimmunological techniques such as chromatography and especially columnchromatography involving magnetic beads. In this approach, the antibodytargeted against the cell surface protein is covalently attached to themagnetic beads, thereby allowing any cells expressing the cell surfaceprotein to be isolated by applying a magnetic field to the cells and themagnetic beads. Bound cells are then removed from the column and grownin microtitre tissue culture plates. Optionally, the cells can be grownin selective media such as G418 supplemented media.

The second high producing cells can be isolated by several techniques.For example, in one particular approach, the cells are grown in thewells for a time sufficient to allow antibody production and secretioninto the culture media. A suitable anti-idiotypic antibody against an Fcportion is coated to the microtitre culture plate wells and the cellculture media is added thereto. The media including high levels of theantibody can be detected by standard techniques such as conventionalsandwhich type assays using a suitable secondary antibody labeled with,e.g., horseradish peroxidase (HRP). Preferred second high producingcells are isolated by identifying high antibody production rates to cellnumber in the plate. Methods for performing this analysis are describedin more detail below and specifically include an ELISA detection format.Preferred are second high producing cells producing from between about0.1 to about 10 micrograms antibody or more per 10⁶ cells over about a24 hour period. A more preferred range is between about 1 to about 5micrograms antibody per 10⁶ cells over about a 24 hour period.

As discussed, additionally preferred are second high producing cellsthat produce from between about 3 to about 40 fold or more of theantibody when compared to the first high producing cells. Antibodyreactivity is a preferred method of making this determination.

Second high expressing cells are further transfected to provide forisolation of cell lines that express more of the protein, e.g., thethird high expressing cells. For example, to increase production of theantibody in the second high cells, the two vectors used to transfect thefirst high expressing cells are again used again to co-transfect thesecond high expressing cells. A preferred transfection method iselectroporation although other transfection methods could be used ifdesired. High producing cells making increased levels of the antibodycan be isolated as discussed above involving magnetic columnchromatography of CD4 expressing cells followed by isolation of highproducing cells. Optionally, the cells can be grown in non-selective orselective media such as G418 supplemented media. Preferred highproducing cells produce maximal levels of antibody as determined byidentifying high antibody production rates to cell number in the plate.

In this example, selected cell lines are preferably serially diluted andgrown by conventional methods in microtitre tissue culture plates. Afterabout a few days up to a few weeks or more, antibody production istested by a suitable immunological technique such as ELISA. Clones withhigh antibody production levels are further amplified. Preferred areclones having from between about 1 to about 100 micrograms antibody ormore per 10⁶ cells over a 24 hour period. A more preferred range is frombetween about 15 to about 50 micrograms antibody per 10⁶ cells over a 24hour period. Additionally preferred are clones that exhibit at leastabout 2 fold up to about 10 fold more antibody production than thesecond high producing cells.

Selected clones satisfying the above criteria are preferably seriallydiluted according to standard methods. After a few days up to a fewweeks or more, single colonies are tested for antibody production by asuitable immunological method such as ELISA. The highest producing clone(third high expressing cell) is selected for preparation of staticcultures as described in detail below. Preferred third high expressingcells produce from about 20 to about 200 micrograms per milliliter ofculture. More preferred third high expressing cells produce from about50 to 150 micorgrams per milliliter of culture with about 100 microgramsper milliliter of culture being generally preferred.

Additionally preferred are third high producing cells produce frombetween about 3 to about 40 fold or more of the antibody when comparedto the second high producing cells as determined by antibody reactivity.

Additionally preferred are third high producing cells produce frombetween about 10 to about 200 fold or more of the antibody when comparedto the first high producing cells as determined by antibody reactivity.

A specific example of a suitable antibody is the anti-TF antibodyencoded by the pJAIgG4TF.A8 vector shown in FIG. 1A.

The high producing cell lines produced by the methods described hereinhave a number of highly useful applications including use in commercial,research and medical settings. For example, it has been found that thehigh producing cells generated by the method are especially amenable tocommercial scale production of the protein sequence. As an illustration,the examples below describe use of a hollow fiber bioreactor to producethe protein sequence on a large-scale (ie. milligram amounts per ml).

In an specific example of the present invention, recombinant mammaliancell lines can be generated that produce high levels of MHC complexesand particularly recombinant MHC single-chain and heterodimericcomplexes. These complexes have been disclosed, e.g., in co-pending U.S.application Ser. No. 08/382,454, filed on Feb. 1, 1995; Ser. No. 08/596,387, filed on Jan. 31, 1996; and PCT application WO 96/04314 publishedon Feb. 15, 1996; the disclosures of which are incorporated herein byreference. Particularly described in the pending U.S. application Ser.Nos. 08/382,454 and 08/596,387 and published PCT application WO 96/04314are a variety of single-chain MHC fusion complexes comprising acovalently linked presenting peptide.

For example, to generate recombinant mammalian cell lines expressing adesired single-chain MHC complex at high levels, a suitable mammalianhost cell such as CHO is transfected once (singly) or at least twice(multiply) with suitable vectors. The vectors each include at least oneselectable marker as defined above and at least one segment encoding thesingle-chain MHC complex. In preferred embodiments, the single-chaincomplex is class II although a class I single-chain complex may bepreferred in some instances. The host cells are preferably transfectedwith one suitable vector encoding the single-chain complex operablylinked to a selectable marker. A variety of transfection methods can beused such as standard calcium phosphate mediated transfection orlipofection techniques. Transfected host cells are then subjected toselective growth conditions so that first high expressing cells can beisolated. Methods for isolating transfected cells have been described ine.g., Sambrook et al., supra; Ausubel et al. supra; and in Wigler PNAS(USA) (1979) 76:1376.

In a more specific method, the vector includes a selectable gene markersuch as a neomycin gene operably linked to the single-chain MHC complex.Host cells are preferably transfected by electroporation and transfectedhost cells expressing the fusion protein sequence are cloned by limitingdilution methods performed in microtitre tissue culture plates. Afterincubating the cells for a day or so up to a few weeks or longer,transfected cells are harvested and diluted in non-selective orselective media such as G418 supplemented media. Supernatants are testedas described above and the examples which follow. High producing clonesare selected and expanded. Selected clones are harvested and grown forseveral days up to a few weeks or more to isolate first high producingcells. Preferred are first high producing cells making from betweenabout 1 to 100 nanograms of the fusion protein per ml of media. Morepreferred are first high producing cells producing from between about 10to 50 nanograms per milliliter.

The first high producing cells making the single-chain MHC fusionprotein are further manipulated as follows. The first high producingcells are further co-transfected, e.g., by electroporation, with twovectors: one that encodes the single-chain fusion protein and anothervector that encodes a suitable cell surface protein such as aglycoprotein and especially CD4. A preferred vector encoding the CD4 isthe commercially available pMACS vector (see below). Transfectants aresubsequently treated with a specific antibody or suitable antigenbinding fragment thereof capable of specifically binding the cellsurface protein. The specific binding can be detected in a variety ofways, however, column chromatography involving magnetic beads is apreferred method. Bound cells are then removed from the column and grownin microtitre tissue culture plates. These second high producing cellscan be grown in non-selective or selective media such as G418supplemented media.

Static cultures of the second high producing cells can be made ifdesired. Preferred are second high producing cells that produce frombetween about 50 to 500 nanograms of the fusion protein per milliliter.More preferred are high producing cells that produce from between about100 to 200 nanograms of the fusion protein per milliliter.

To further increase production of the single-chain MHC complex, thesecond high producing cells can be additionally transfected. In oneapproach, the second high producing cells are co-transfected with amixture of the vector encoding the single-chain MHC fusion protein andanother vector carrying a selectable nucleic acid sequence that confersresistance to a drug such as puromycin or other suitable drug. Theco-transfection can be performed by any suitable method includingelectroporation if desired. After incubation for a few days up to a fewweeks or more, cells are harvested and resuspended in media supplementedwith puromycin. After visualization of resistant colonies, culture mediacan be tested for production of the fusion protein, e.g, by ELISA.Static cultures can be made from clones producing high levels of theprotein. Preferred third high producing cells produce from between about500 to about 5000 nanograms per milliliter of the fusion protein. Morepreferred are third high producing cells that produce from between about1000 to 2000 nanograms per milliliter of the protein.

If desired, suitable third high producing cells can be further subclonedto improve recombinant protein expression. A preferred method is limiteddilution cloning. Additionally preferred are clones of the third highproducing cells producing from between about 100 to about 1000 nanogramsof the fusion protein per milliliter over 24 hours. Particularlypreferred are the clones producing from between about 200 to 500nanograms of the fusion protein per milliliter over 24 hours.

An example of a vector encoding a single-chain MHC complex is the pDRHKvector shown in FIGS. 1B. That vector encodes a sc-DR2/MBP single-chainclass II MHC complex.

The present methods can be varied to suit intended use. For example, theinvention specifically encompasses the following embodiments: 1)transfection with an expression vector carrying a drug resistance gene,2) co-transfection with an expression vector and a vector thattransiently expresses a cell surface marker, and 3) co-transfection withan expression vector and a vector carrying a different drug resistancegene. The cell selection procedures discussed herein can be used toimplement these specific strategies. The invention can also be used tore-transfect cell with expression vectors encoding specific recombinantproteins of interest such as when an expression vector encodes twopolypeptide sequences (immunoglobin heavy and light chains). Preferredexpression involves coordinate expression of both polypeptide chains.These specific methods and others discussed herein find a variety ofuses including facilitating selection and screening of cells.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not to be construed as a limitation thereof.

Example I Retransfection of CHO Cells for High Level Production ofRecombinant Anti-tissue Factor (TF) Antibody

1. Vector Characterization

A vector referred to as pJAIgG4TF.A8 (FIG. 1A) was constructed toexpress chimeric anti-tissue factor (TF) heavy and light immunoglobulinchains in mammalian cells. Initial transient transfection experimentswere carried out to test antibody expression from the pJAIgG4TF.A8vector. COS cells were transiently transfected using the Qiagenlipofectin reagent. Briefly, 2.5×10⁵ cell/well were seeded in a 6 wellplate and incubated for 24 hours at 37° C. Two micrograms ofpJAIgG4TF.A8 DNA were mixed with 100 μl of IMDM. To make the lipidcomplex, 10 μl of lipids was added to the DNA solution. The mixture wasvortexed and incubated at room temperature for 5 minutes. While DNA wasforming the lipid complex, the cells were washed with PBS. The DNA-lipidmixture was mixed with 600 μl of 10%SSM [10% fetal bovine serum (FBS)supplemented CellGro IMDM (MediaTech) media] and added to the washedcells. The cells were incubated with lipid complexes for 3 hours at 37°C. and 10% CO₂. The transfected cells were washed with PBS, fed 2 ml of10% SSM and incubated for 72 hours at 37° C. and 10% CO₂. The culturesupernatant was tested for antibody production.

2. ELISA to Test Antibody Production

To detect the presence of the human IgG4 antibody, a humanHC/kappa-specific sandwich ELISA was developed. Briefly, Maxisorp 96well plates (NUNC) were coated with 100 μl of 1 μg/ml Goat anti-HumanIgG-Fc (Fab) in R5 buffer (10 mM Tris-HCI, pH 8.5) and incubated at 4°C. overnight. The wells were then washed with R55 buffer (2 mMImidazole, 7.5 mM NaCI, 0.02% Tween-20) once, covered with a plasticfilm and stored at 4° C. until used. To assay antibody production, theR55 was removed and 100 μl of transfectant supernatant was added to thecoated wells. After 30 minutes at 37° C., the wells were washed 6 timeswith R55 buffer and 100 μl of a 1:800 dilution (in PBS containing 10%FBS) of an anti-human Kappa chain-HRP antibody (Southern Biotech) wasadded. The plates were then incubated at 37° C. and washed 6 times with400 μl of R55 buffer. To detect the presence of the probe antibody, 100μl of 1×ABTS substrate (Kirkegaard & Perry Labs) was added for 4 minutesand followed by 100 μl of ABTS quench buffer (Kirkegaard & Perry Labs).Absorbance was read at 405 nm. Purified human IgG4 protein served as apositive control. The results from such an assay showed that theanti-TFIgG4 antibody was produced by COS cell transiently tranfected bythe pJAIgG4TF.A8 vector.

3. ELISA to Test Antibody Specificity

A second sandwich ELISA was developed to specifically detect antibodybinding to human tissue factor. Maxisorp 96 well plates (NUNC) werecoated overnight at 4° C. with 100 μl of 500 ng/ml recombinant human -TFin R65 buffer (100 mM Sodium Bicarbonate, pH 8.2). The next day theplates were washed with R55 buffer, covered and stored at 4° C. untilused. To detect anti-TF antibody production, the R55 was removed and 100μl of transfectant supernatant was added to the coated wells. After 30minutes at 37° C., the wells were wash 6 times with R55 buffer and 100μl of a 1:800 dilution (in PBS containing 10% FBS) of an anti-humanKappa chain-HRP (Southern Biotech) was added. The plates were thenincubated at 37 ° C. and washed 6 times with 400 pi of R55 buffer. Todetect the presence of the probe antibody, 100 μl of 1×ABTS substratewas added for 4 minutes and followed by 100 μl of ABTS quench buffer.Absorbance was read at 405 nm. Purified mouse H36.D2 anti-TF proteinserved as a positive control. The results from such an assay showed thatthe anti-TF IgG4 antibody was produced by COS cell transientlytranfected by the pJAIgG4TF.A8.

4. Initial Stable Transfection of CHO Cells

To generate a stable cell line expressing the recombinant anti-TFantibody, CHO.K1 cells were transfected with the pJAIgG4TF.A8 vector.Briefly, 100 μg of pJAIgG4TF.A8 DNA was linearized by digesting withPvul at 37° C. for about 4 hours. CHO.K1 cells (ATCC CCL-61) werediluted to a concentration of 1.25×10⁷ cell/ml. A volume of 800 μl ofcells was added to an 0.4 cm electroporation cuvette and incubated onice for 10 minutes. Twenty five micrograms of pJAIgG4TF.A8 DNA wereadded, mixed with the cells, and incubate for 10 minutes. The cells werethen electroporated at 960 μF and 250 V. Following a 10 minuteincubation on ice, the cell suspension was added to a T25 flask with 10ml 10%SSM and incubated overnight at 37° C. in 10% CO₂. Twenty-fourhours later, the cells were harvested by incubation with trypsin-PBS,resuspended in PBS and diluted in G418 supplemented media at 1: 9, 1:27and 1:81. The transfected cells were plated at 100 μl/well in 96 wellplates and incubated at 37° C. in 10% CO₂.

Culture supernatant from the transfected cells was tested for antibodyproduction as described above. Positive clones were selected andexpanded. Clone H9 from row H column 9 of the 1:27 dilution plate wasselected as a high antibody level producer.

This cell line was cloned by limiting dilution. Briefly, the selectedclone was diluted to 1000 cell/ml in PBS. A series of 1:10 dilutions in10% SSM were made to obtained 1 cell/ml. From these dilutions, 100μl/well were plated in 96 well flat bottom plates. These plates wereincubated at 37° C. in 10% CO₂. After three days of incubation, 100μl/well of 10%SSM was added to each well. Three weeks later, singlecolonies started to become apparent. Only single colonies were testedfor antibody production by ELISA. Clones with high levels of antibodyproduction were amplified and selected. Clone H9g12 was selected ashighest producer and analyzed for antibody production in static culture.

The selected clones were harvested by treatment with trypsin,resuspended at a concentration of 1×10⁵ cell/ml in 10 ml of 10% SSMmedia and added to a T-25 tissue culture flask. The cells were incubateat 37° C. in 10% CO2 for 21 days or until 80% cell death. The cellsuspension was centrifuged and the supernatant was tested for Abproduction by ELISA assay. The maximum antibody production of the H9g12clone was 3.5 μg/ml.

5. Stable Re-transfection of the CHO-H9g12 Cell Line

This method was developed to add more copies of the genes of interest tothe genome of a stable Ab-producing cell line. To proceed with thismethod, clone H9g12 was co-transfected with a mixture of the anti-humanTF mega vector pJAIgG4TF.A8 and pMACS. The pMACS vector allows for thetransient expression of a membrane bound CD4 protein. To co-transfectthe H9g2 cell line, 800 μl of a 1.25×10⁷ cell/ml suspension was added toa 0.4 cm electroporation cuvette and incubated on ice for 10 minutes. A3: 1 molar ratio of pJAIgG4TF.A8 and pMACS DNA (40 μl of 1 μg/mlPvul-linearized pJAlgG4TF.A8 and 5 μl of 1 μg/ml supercoiled pMACS) wasadded to the cells. After incubating on ice for 10 minutes, the cellswere electroporated at 960 μF and 250 V. The cells were incubate at 37°C. for 10 minutes, diluted to 10 ml 10% SSM in a T25 flask and incubated72 hours at 37° C. in 10% CO₂ to allow the transient expression of theCD4 protein. At this time, the cells were treated with 5 ml of PBE (EDTAin PBS solution) at room temp until they detached. Cells were wash once,resuspended in 380 μl of PBE and labeled with 80 μl of an antibodyspecific to the cell surface-expressed human CD4 molecule. This antibodywas covalently bound to a magnetic bead. Following incubation for 15 to20 minutes at 4° C., the antibody labeled-cells were applied to amagnetic column. Transfected cells that express the CD4 on theirsurfaces are expected to bind the magnetic column. The untransfectedcells were washed through the column with 1 ml PBE. The bound cells werethen eluted from column by removing column from magnetic field andpassing 1 ml of PBE through the column. CD4-expressing cells werediluted in 199 ml of 10%SSM supplemented with G418 (1.5 mg/ml) andplated in 96 well flat bottom plate. The plates were incubated at 37° C.in 10% CO₂.

To test for antibody production, Maxisorp 96 well plates (NUNC) werecoated with 100 μl of 1 μg/ml Goat anti-Human IgG-Fc (Fab) (Pierce) asdescribed above. The media from re-transfected cells was changed 24hours prior to testing. Five microliters of the transfectant 24 hoursupernatant were added to 95 μg of 10% FBS-PBS. The diluted supernatantwas added to the coated wells and was incubated for 60 minutes at 37° C.The wells were washed 6 times with R55 buffer and an anti-human kappachain antibody-HRP (Southern Biotech) was used to detected recombinantantibody present in the supernatant as described above. Purified humanIgG4 protein (Biodesign) served as a positive control and was used toestablish a standard curve for antibody concentration.

The clone selection was accomplished by comparing antibody productionrates to cell number. Briefly, the high producing clones were trypsintreated and counted. Using the calculated antibody production rate andthe number of cells, values for the μg antibody/10⁶ cells /24 hour weredetermined. High producer clones were amplified to 24 well/plates andexpanded. The clone selected was named 3D2.

For limited dilution cloning, the selected clones were serially dilutedin 10% SSM, seeded in 96 well flat bottom plates, incubated as describedabove. After three weeks, single colonies were tested for antibodyproduction by ELISA. Clones with high levels of antibody production wereamplified and selected. Clone 3D2A9 showed 2.58 μg antibody /106 cells/24 hour and was selected as the best clone. Analysis of antibodyproduction in static culture was initiated for this clone.

Static cultures of the 3D2A9 clone were initiated by adding aconcentration of 2×10⁵ cells/ml in 10 ml of 10%SSM media to a T-25tissue culture flask. The flasks were incubated at 37° C. in 10% CO2 for21 day or until 80% cell death. The cell suspension was centrifuged andsupernatant was tested for antibody production by ELISA. The maximumantibody production of the 3D2A9 clone was 21 μg/ml. This productionlevel was 7 fold higher than the first transfection clone.

6. Third Stable Transfection

To further increase the antibody production level of the transfectedcells, the clone 3D2A9 was again co-transfected with a mix of theanti-human TF mega vector pJAIgG4TF.A8 and pMACS. To cotransfect thiscell line, 800 μl of a 1.25×10⁷ cell/ml suspension was added to a 0.4 cmelectroporation cuvette and incubated on ice for 10 minutes. A 3:1 molarratio of pJAlgG4TF.A8 and pMACS DNA (50 μl of 1 μg/ml Pvul-linearizedpJAIgG4TF.A8 and 5 μl of 1 μg/ml supercoiled pMACS) was added to thecells. After incubating on ice for 10 minutes, the cells wereelectroporated at 960 μF and 250 V. The cells were incubate at 37° C.for 10 minutes, added to 10 ml 10%SSM in a T25 flask and incubated 48hours at 37° C. in 10% CO₂ to allow the transient expression of the CD4protein. At this time, the transfected cells were labeled with theanti-CD4 magnetic beads and selected on the magnetic column as describedabove. CD4-expressing cells were diluted in 200 ml of 10%SSMsupplemented with G418 (1.5 mg/ml). The cell suspension was plated in 96well flat bottom plate. The plates were incubated at 37° C. in 10% CO₂for approximately three weeks.

Maxisorp 96 well plates (NUNC) were coated with 100 μl of 1 μg/ml Goatanti-Human IgG-Fc (Fab) (Pierce) in R5 as described above. To assayantibody production rates, media from re-transfected cells was changed24 hours prior to testing. Five microliters of the transfectant 24 hoursupernatant were added to 95 μl of 10% FBS-PBS. The diluted supernatantwas added to the coated wells and was incubated for 60 minutes at 37° C.The wells were washed 6 times with R55 buffer and an anti-human Kappachain antibody-HRP (Southern Biotech) was used to detected recombinantantibody present in the supernatant as described above. A purifiedanti-TF chimeric antibody served as a positive control and was used toestablish a standard curve for antibody concentration. Results from thistest allowed the selection of high antibody producing clones that werethen amplified to a 12 well plate. These clones were grown to 80% of thewell and 24 hour supernatants was tested. A 1:100 dilution of the 24 hrculture supernatant was tested by ELISA. Cells were also counted and μg/10⁶ cells/24 hour antibody production values were determine. Highestantibody producing mother clones A9B 11 and A9F 12 was selected based onthese values.

Selected clones were serially diluted in 10% SSM, seeded in 96 well flatbottom plates, incubated as described above. After three weeks, singlecolonies were tested for antibody production by ELISA. Clones with ahigh antibody production level were amplified. The primary clonesA9F12C2 and A9AIIBS were found to have the highest clones with anantibody production rate of 30 to 40 μg /10 ⁶cells /24 hours. The cloneswere expanded for static cultures.

Static cultures of the A9F12C2 clone were initiated by adding aconcentration of 2×10⁵ cells/ml in 10 ml of 10% SSM media to a T-25tissue culture flask. The flasks were incubated at 37° C. in 10% CO₂ for21 day or until 80% cell death. The cell suspension was centrifuged andsupernatant was tested for Ab production by ELISA The maximum antibodyproduction of the A9F12C2 clone was 52 μg/ml. This production level was2 fold higher than the second transfection clone.

The selected clone A9F12C12 was serially diluted in 10% SSM, seeded in96 well flat bottom plates, incubated as described above. After threeweeks, single colonies were tested for antibody production by ELISA. Thesecondary clone A9F12C2E7 was found to have the highest antibodyproduction rate and was expanded for static cultures. These staticcultures were carried out as described above.

To determine the antibody concentration in the static culturesupernatants, 5 μl of A9F12C2E7 cell supernatant was added to 4,995 μlof 10% PBS. This diluted supernatant was assayed for the chimericantibody by the antibody ELISA described above. As a positive control,the purified anti-TF chimeric antibody was used. The results from thistest indicated that the A9F12C2E7 secondary clone had a maximum antibodyproduction level of 52.6 μg/ml in static culture.

The selected clone A9F12C12E7 was serially diluted in 10% SSM, seeded in96 well flat bottom plates, incubated as described above. After threeweeks, single colonies were tested for antibody production by ELISA. Thetertiary clone A9F12C2E7B4 was selected as the highest producer and wasexpanded for static culture. These cultures were initiated as previouslydescribed.

To determine the antibody concentration in the static culturesupernatants, 5 μl of A9F12C2E7 cell supernatant was added to 7,995 μlof 10% PBS. This diluted supernatant was assayed for the chimericantibody by the antibody ELISA described above. As a positive control,the in-house purified anti-TF chimeric antibody was used. The maximumantibody production for clone A9F12C2E7B4 was 102.3 μl/ml (FIG. 2). Thecell line was amplified and frozen. Fifty vials at a concentration of1×10⁶ cells/ml were made as an initial seed cell bank and were stored inliquid nitrogen. Name designation for cell line is cH36 (chimeric H36).

4. Bioreactor Injection

A hollow fiber bioreactor was set up as indicated in the manufacturerinstructions. Selected clone was resuspended at 1×10⁸ cells/ml in thecorrect volume of 30%SSM and injected into the bioreactor. Thebioreactor was then run as specified by manufacturer instructions.Maximum production was tested by ELISA as described above. The firsttransfection primary clone H9g12 produced 70 μg antibody/ml at maximumas compared to the third transfection primary clone A11B5 which produced1,100 μg antibody/ml (FIG. 3).

Example 2 Generation of Cell Lines for Production of Recombinant HLA-DR2MHC Class II Molecule

1. Vector Characterization

A vector referred to as pDRHK (FIG. 1B) was constructed for mammalianexpression of soluble single-chain HLA-DR2/MBP molecule fused to thehuman immunoglobulin kappa constant domain. Initial transienttransfection experiments were carried out to test protein expressionfrom the pDRHK vector. COS cells were transiently transfected using theQiagen lipofectin reagent. Briefly, 2.5×10⁵ cell/well were seeded in a 6well plate and incubated for 24 hours at 37° C. Two micrograms of pDRHKDNA were mixed with 100 μl of IMDM. To make the lipid complex, 10 μl oflipids was added to the DNA solution. The mixture was vortexed andincubated at room temperature for 5 minutes. While DNA was forming thelipid complex, the cells were washed with PBS. The DNA-lipid mixture wasmixed with 600 μl of 10%SSM and added to the washed cells. The cellswere incubated with lipid complexes for 3 hours at 37° C. and 10% CO₂.The transfected cells were washed with PBS, fed 2 ml of 10% SSM andincubated for 72 hours at 37° C. and 10% CO₂. The supernatant was testedfor recombinant DR2/MBP-CK production.

2. ELISA to Detect Sc- DR2/MBP-CK Production

To detect the presence of the sc-DRZ/MBP-C6, a human 6/HLA-DR-specificsandwich ELISA was developed. Briefly, Maxisorp 96 well plates (NUNC)were coated with 100 μl of 1 μg/ml Goat anti-human IgG-kappa in PBS andincubated at 4° C. overnight. The wells were then washed with R55 bufferonce, covered with a plastic film and stored at 4° C. until used. Toassay recombinant protein production, the R55 was removed and 100 μl oftransfectant supernatant was added to the coated wells. After 60 minutesat 37° C., the wells were wash 6 times with R55 buffer and 100 μl of a1:1000 dilution (in PBS containing 10% FBS) of an anti HLA-DR antibodyL243 conjugated to HRP (Anergen, ATCC HB-55) was added. The plates werethen incubated at 37° C. and washed 6 times with 400 μl of R55 buffer.To detect the presence of the probe antibody, 100 μl of 1× ABTSsubstrate was added for 5 minutes and followed by 100 μl of ABTS quenchbuffer. Absorbance was read at 405 nm. The results from such an assayshowed that the scDR2/MBP-C κ fusion protein was produced by COS celltransiently tranfected by the pDRHK vector.

3. Initial Stable Transfection

To generate a stable cell line expressing the sc-DR2/MBP-Cκ fusionprotein, CHO.K1 cells were transfected with the pDRHK vector. Briefly,100 μg of pDRHK DNA was linearized by digesting with Pvul at 37° C. forabout 4 hours. CHO.K1 cells (ATCC CCL-61) were diluted to aconcentration of 1.25×10⁷ cell/ml. A volume of 800 μl of cells was addedto an 0.4 cm electroporation cuvette and incubated on ice for 10minutes. Twenty micrograms of pDRHK DNA were added, mixed with thecells, and incubate for 10 minutes. The cells were then electroporatedat 960 μF and 250 V. Following a 10 minute incubation on ice, the cellsuspension was added to a T25 flask with 10 ml 10%SSM and incubatedovernight at 37° C. in 10% CO₂. Twenty-four hours later, the cells wereharvested by incubation with trypsin-PBS, resuspended in PBS and dilutedin G418 supplemented media at 1:9, 1:27 and 1:81. The transfected cellswere plated at 100 μl/well in 96 well plates and incubated at 37° C. in10% CO₂.

Supernatant from cells from different 96 well plates were tested asdescribed above. Positives clones were selected and expanded. Clone A5from row A column 5 of the 1:27 plate was selected as a high producer.This cell line was subdloned by limiting dilution as described above.After screening, clone A5B4 was selected as highest producing cell lineand static culture was initiated.

The selected clones were harvested by treatment with trypsin,resuspended at a concentration of 1×10⁵ cells/ml in 10 ml of 10% SSMmedia and added to a T-25 tissue culture flask. The cells were incubateat 37° C. in 10% CO₂ for 21 days or until 80% cell death. The cellsuspension was centrifuged and the supernatant was tested forrecombinant DR2 production by ELISA assay. Recombinant sc-DR2/MBP-C6fusion production by the A5B4 clone was 20 ng/ml.

4. Stable Re-transfection

To proceed with this method, clone A5B4 is co-transfected with a mix ofpDRHK and pMACS. A volume of 800 μl of a 1.25×10⁷ cell/ml suspension wasadded to a 0.4 cm electroporation cuvette an incubated on ice for 10minutes. A 3:1 molar ratio of pDRHK and pMACS DNA (30 μl of 1 μg/ml Pvullinearized pDRHK and 5 μl of 1 μg/ml supercoiled pMACS) was added to thecells. After incubating on ice for 10 minutes, the cells wereelectroporated at 960 μF. and 250 V. The cells were incubate at 37° C.for 10 minutes, diluted to 10 ml 10%SSM in a T25 flask and incubated 72hours at 37° C. in 10% CO₂ to allow the transient expression of the CD4protein. At this time, the cells were treated with 5 ml of PBE at roomtemperature until they detached. Cells were wash once, resuspended in380 μl of PBE and labeled with 80 μl of an antibody specific to the cellsurface-expressed human CD4 molecule. This antibody was covalently boundto a magnetic bead. Following incubation for 15 to 20 minutes at 4° C.,the antibody labeled-cells were applied to a magnetic column.Transfected cells that express the CD4 on their surfaces are expected tobind the magnetic column. The untransfected cells were washed throughthe column with 1 ml PBE. The bound cells were then eluted from columnby removing column from magnetic field and passing 1 ml of PBE throughthe column. CD4-expressing cells were diluted in 199 ml of 10% SSMsupplemented with G418 (1.5 mg/ml) and plated in 96 well flat bottomplate. The plates were incubated at 37° C. in 10% CO₂.

After 3 weeks, the supernatant from different 96 well plates were testedfor recombinant protein as described above. Positive clones wereselected and expanded. Clone DR2² was selected as the highest producer.This cell line was frozen.

A vial of DR2² clone was quick thawed in a 37° C. water bath and grownto 95% viability. The cells were serially diluted for limited dilutioncloning as described above. After three weeks of growth, single colonieswere tested for sc-DR2-Cκ production by ELISA. The clones with thehighest production levels were amplified and selected. Clone DR2²-H4 wasidentified as the best producer. Static cultures were carried out asdescribed above and recombinant protein production from the DR2²-H4clone was found to be 100 ng/ml.

4. Third Stable Transfection

To further increase recombinant protein production, primary cloneDR2²-H4 was co-transfected with a mixture of pDRHK and pPUR (Clonetech),a vector carrying a gene that confers resistance to puromycin. Briefly,800 μl of a 1.25×10⁷ cell/ml suspension was added to a 0.4 cmelectroporation cuvette and incubated on ice for 10 minutes. A 3:1 molarratio of pDRHK and pMACS DNA (25 pl of I μg /ml Pvullinearized pDRHK and5 μl of 1 μg/ml Pvul-linearized pPUR) was added to the cells. Afterincubating on ice for 10 minutes, the cells were electroporated at 960μF and 250 V. The cells were incubate at 37° C. for minutes, added to 10ml 10%SSM in a T25 flask and incubated 48 hours at 37° C. in 10% CO₂.The cells were then harvested by trypsin treatment, centrifuged andresuspended in 10%SSM containing G418 (1.5 mg/ml) and Puromycin (20μg/ml). The cells were plated in 96 well flat bottom plates andincubated at 37° C. in 10% CO₂. After colonies were apparent, theculture media was tested for recombinant protein. Clones A9 and B9 werefound to be the highest producers and were tested in static cultures asdescribed above. The static culture recombinant protein production was1,800 ng/ml for A9 and 2,108 for B9 ( FIG. 4).

These cell lines were subcloned by limited dilution cloning and testedfor recombinant protein production. Primary clones A9D5, A9G4, A9C7,B9H3, B9G4, and B9H5 were identified as having scDR2/MBP-C_(κ)production rates of 200 to 300 ng/ml in 24 hours. Recombinant proteinproduction by these clones in static cultures was carried out asdescribed above.

The invention has been described in detail with reference to preferredembodiments thereof However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements within the spirit and scope of theinvention.

What is claimed is:
 1. A method for producing a cell line featuringexpression of an amino acid sequence at high levels, the methodcomprising: a) introducing into host cells twice or more a first vectorcomprising first selectable sequence operably linked to a segmentencoding at least one copy of the amino acid sequence, b) culturing thehost cells under conditions conductive to selecting the first vector;and c) isolating cells expressing the amino acid sequence at a firstexpression level to produce a cell line (first high expressing cells).2. A method for producing a cell line featuring expression of an aminoacid sequence at high levels, the method comprising: a) introducing intohost cells once or more than once a first vector comprising firstselectable sequence operably linked to a segment encoding at least onecopy of the amino acid sequence, b) culturing the host cells underconditions conductive to selecting the first vector, c) isolating cellsexpressing the amino acid sequence at a first expression level toproduce a cell line (first high expressing cells), wherein the methodfurther comprises: d) introducing into the first high expressing cellsonce or more than once a second vector encoding the amino acid sequence;and e) subjecting the cells to conditions conducive to expressing theamino acid sequence at a second expression level higher than the firstexpression level, and isolating cells expressing the amino acid sequenceat the second expression level to produce a cell line (second highexpressing cells).
 3. The method of claim 1 or 2, wherein the isolationfurther comprises selecting cells expressing a drug resistance marker.4. The method of claim 1 or 2, wherein the isolation further comprisesselecting cells expressing a cell-surface marker.
 5. The method of claim4, wherein expression of the cell-surface marker is transient.
 6. Themethod of claim 3, wherein the drug resistance marker is aminoglycosideantibiotic (G418), hygromycin B (hmb), puromycin, or neomycin.
 7. Themethod of claim 1, wherein the method further comprises selecting cellsexpressing a cell surface marker.
 8. A method for producing a cell linefeaturing increased expression of an amino acid sequence, the methodcomprising: a) introducing into host cells a first vector comprising afirst selectable sequence operably linked to a segment encoding at leastone copy of the amino acid sequence, b) culturing the host cells underconditions conducive to selecting the first vector, c) isolating cellsexpressing the amino acid sequence at a first expression level; and d)subjecting the cells to conditions conducive to expressing the aminoacid sequence at a second expression level higher than the firstexpression level to produce the cell line, wherein the isolation furthercomprises selecting cells expressing a cell-surface marker.
 9. Themethod of claim 8, wherein expression of the cell-surface marker istransient.
 10. The method of claim 8, wherein step c) of the methodfurther comprises selecting cells expressing a drug resistance marker.11. The method of claim 10, wherein the drug resistance marker isaminoglycoside antibiotic (G418), hygromycin B (hmb), puromycin, orneomycin.
 12. A method for producing a genetically stable cell linefeaturing increased expression of an amino acid sequence, the methodcomprising: a) introducing into host cells a first vector comprising afirst selectable sequence operably linked to a segment encoding at leastone copy of the amino acid sequence, b) culturing the host cells underconditions conducive to selecting the first vector, c) isolating cellsexpressing the amino acid sequence at a first expression level, d)subjecting the cells to conditions conducive to expressing the aminoacid sequence at a second expression level higher than the firstexpression level; and e) detecting the first vector within the genome ofthe cells to produce the genetically stable cell line, wherein themethod further comprises selecting cells expressing a cell-surfacemarker.
 13. A cell line produced by the method of claim 1, 8 or
 12. 14.The method of claim 1 or 12, wherein the first vector is introduced intothe host cells more than once and between each vector introduction, thehost cells are cultured under conditions conducive to selecting thefirst vector.
 15. The method of claim 12, wherein the isolation furthercomprises selecting cells expressing a cell-surface marker.
 16. Themethod of claim 12, wherein step c) of the method further comprisesintroducing into the cells expressing at the first expression level asecond vector encoding at least one copy of the amino acid sequence. 17.The method of claim 12, wherein step c) of the method further comprisesselecting cells expressing a drug resistance marker.
 18. The method ofclaim 17, wherein the drug resistance marker is aminoglycosideantibiotic (G418), hygromycin B (hmb), puromycin, or neomycin.
 19. Amethod for producing a multi-subunit protein, the method comprising: a)introducing into host cells a first vector comprising a first selectablesequence operably linked to a segment encoding at least one copy of afirst subunit of the protein, b) culturing the host cells underconditions conducive to selecting the first vector, c) isolating cellsexpressing the amino acid sequence at a first expression level (firsthigh expressing cells), d) introducing into the cells expressing at thefirst expression level a second vector encoding at least one copy of asecond subunit of the protein, e) subjecting the cells to conditionsconducive to expressing the amino acid sequence at a second expressionlevel higher than the first expression level; and f) isolating the cellsexpressing the amino acid sequence at the second expression level(second high expressing cells) to produce the multi-subunit protein. 20.The method of claim 19, wherein the isolation further comprisesselecting cells expressing a cell-surface marker.
 21. The method ofclaim 19, wherein step c) of the method further comprises selectingcells expressing a drug resistance marker.
 22. The method of claim 21,wherein the drug resistance marker is aminoglycoside antibiotic (G418),hygromycin B (hmb), puromycin, or neomycin.